1. Field of the Invention
The present invention relates to a biopolymer crystal mounting device to be used for taking a biopolymer crystal out of a solution and mounting it at the time of providing the biopolymer crystal having been grown in the solution containing a biopolymer typically represented by a protein for use in crystallography, and to a method of manufacturing such device for mounting a crystal thereof.
2. Description of the Related Art
Making clear a three-dimensional structure of a biopolymer typically represented by a protein, not only enables to elucidate physiological functions of the biopolymer in vivo, but also is extremely useful for the purpose of advancing a rational development of drugs (drug design). As a practical method for analyzing a three-dimensional structure of the biopolymer, there have been an NMR (nuclear magnetic resonance) and an X-ray crystallography. From the viewpoint of analysis, the X-ray crystallography in which there is no restriction on a molecular weight of a biopolymer is considered especially effective from now on. In this X-ray crystallography, however, a single biopolymer crystal to be provided for use in the analysis has to be prepared. Moreover, to improve a resolution of the structure analysis, it is necessary to form a single crystal having a high crystallinity.
As a method for preparing a single biopolymer crystal such as protein, a vapor diffusion method is popularly employed these days. In a sitting drop vapor diffusion method, as shown in FIG. 8, a small volume of aqueous solution 1 of about 1 μl containing a protein is dropped into a concave part 3 of a well solution retention plate 2 (in a hanging drop vapor diffusion method, a small volume of aqueous solution 1 containing a protein is dropped onto a surface being a lower face side of a cover glass 6 and adhered thereto). Further, a precipitant 5 is contained in an inner bottom portion of a container-shaped concave part (well) 4, and a top opening of the container-shaped concave part 4 is closed tightly with the cover glass 6. Thus the aqueous solution 1 containing a protein comes to be in a supersaturated state due to evaporation of moisture in the course of time, and eventually a crystal 7 of the protein is precipitated in the foregoing aqueous solution 1.
After having obtained the crystal 7 of a protein, the crystal 7 is provided for use in crystallography. In the crystallography, a diffraction intensity of the crystal is measured using an X-ray diffraction measurement apparatus. Therefore it is required to get the crystal 7, which has grown within the aqueous solution 1, out of the concave part 3 of the well solution retention plate 2. Then, the crystal having been taken out is frozen with, e.g., liquid nitrogen, and thereafter this frozen crystal is irradiated with X-ray to collect X-ray diffraction data. A crystal of protein, however, is extremely soft inherently, and possesses brittle properties. In case where the taking out a crystal and treatments in each step of freezing thereof are defective when collecting X-ray diffraction data, the crystal having been grown all the way will be broken, or minute cracks will be made within the crystal, resulting in a trouble that data cannot be collected.
For that purpose, the following method has been widely employed. In this method, first as shown in FIG. 9(a), a tool 9 of such a structure as shown in FIG. 9(b) is fabricated by inserting a ring made of nylon, what is called as nylon loop (diameter thereof is approximately 1 mm or not more than 1 mm), into a support 8b made of metal, what is called as micro-tube, and securing it. Further, in the method, with the use of the mentioned tool 9, a biopolymer crystal is taken out of a solution, the crystal that is mounted on the ring 8a of the tool 9 is frozen, and thereafter X-ray diffraction data is collected. This method is described in more detail referring to FIG. 10. First, as shown in FIG. 10(a), the crystal 7 of a protein is picked up from the concave part 3 along with a part of the aqueous solution 1 with the use of the tool 9. Normally this operation is carried out manually under a microscope. Then, as shown in FIG. 10(b) , the crystal 7 of a protein is retained along with the aqueous solution 1 in the ring 8a of the tool 9. Next, though not shown, the ring 8a of the tool 9 on which the crystal 7 is retained is dipped into a defrost (i.e., anti-freezing agent), whereby, as shown in FIG. 10(c), moisture of the aqueous solution 1, which is retained within the ring 8a of the tool 9, is substituted with a defrost 1′. Thereafter, as shown in FIG. 10(d), to protect the crystal 7, a liquid nitrogen gas stream F is sprayed obliquely from above to the crystal 7 retained within the ring 8a of the tool 9, and the crystal within the ring 8a is made to freeze. Subsequently, the crystal 7 of a protein retained in the ring 8a of the tool 9 is irradiated with X-ray from the side, and the measurement of diffraction intensity is carried out (see, for example, S. Ohno, S. Yano (eds.), (ed, Chemical Society of Japan) “Basic Course 12 for Chemists, X-ray Structure Analysis” Asakura Co., pp. 102-108, March, 1999).
As described above, according to the conventional method of using the tool 9 that is fabricated by securing a ring (nylon loop) 8a to a support (micro-tube) 8b, there exist several problems in practical use as described hereinafter.
(1) The tool 9 shown in FIG. 9(b) is normally manufactured manually. That is, as shown in FIG. 9(a), the tool 9 is manufactured by first tying up nylon fibers into a bundle to form into a ring shape and twisting both end portions of the nylon fiber bundle with each other to fabricate a nylon loop, and thereafter inserting the twisted portion of the nylon loop into the micro-tube and securing it thereto with an adhesive or the like. Since the tool 9 is manually fabricated in such a way, the fabrication thereof is extremely inefficient, and requires much time. Moreover, a burden of labors on a worker is large in the manual fabrication. Furthermore, a loop may be manufactured to be inaccurate in diameter resulting in the reduction in yield. A further problem exists in that the tool 9 cannot be mass-produced since the tool 9 is fabricated in manual works.
(2) Since fibers forming a nylon loop is approximately 10 μm to 20 μm in diameter, as shown in FIG. 10(d), the nylon loop is vibrated due to the wind pressure of a liquid nitrogen gas stream F when a crystal is retained in the ring 8a of the tool 9 and the crystal is frozen, leading to a further problem that diffraction data cannot be collected.